Method for the production of magnesium pyridoxal-5&#39;-phosphate glutamate and intermediate products obtained thereby

ABSTRACT

The invention relates to a method for the production of magnesium pyridoxal-5&#39;-phosphate glutamate, in which pyridoxine or an acid addition salt thereof is oxidised with manganese (IV) oxide to pyridoxal; pyridoxal is reacted with p-phenetidine under formation of the Schiff&#39;s base p-phenetidyl-pyridoxal; p-phenedityl-pyridoxal is selectively phosphorylated on the 5&#39;-hydroxymethyl group under formation of p-phenetidyl-pyridoxal-5&#39;-phosphate; p-phenetidyl-pyridoxal-5&#39;-phosphate is hydrolysed under formation of an alkali metal salt of pyridoxal-5&#39;-phosphate; the alkali metal ions are removed in order to obtain pyridoxal-5&#39;-phosphate; pyridoxal-5&#39;-phosphate is reacted with a reaction product of a magnesium alcoholate and L-glutarnic acid; and the formed magnesium pyridoxal-5&#39;-phosphate glutamate is isolated. Furthermore, the invention relates to intermediate products obtained with this method.

CROSS-REFERENCE

This application is a 371 of PCT/EP96/03749 filed Aug. 26, 1996.

The invention relates to the method for the production of magnesiumpyridoxal-5'-phosphate glutamate. The invention especially relates to anew method for the production of magnesium pyridoxal-5'-phosphateglutamate starting from an acid addition salt of pyridoxine, such aspyridoxine hydrochloride, in which pyridoxal-5'-phosphate is obtained asan intermediate product.

Magnesium pyridoxal-5'-phosphate glutamate is known from DE 24 61 742.In this publication, it is proposed as a medicament for prophylaxis andtherapy of metabolic disturbances, especially for influencing the lipidand cholesterol state. DE 40 16 963 relates to the use of magnesiumpyridoxal-51'-phosphate glutamate for the reduction of LDL-boundperoxides and for prevention of vascular damage.

Considering the importance of magnesium pyridoxal-5'-phosphate glutamateas a medicament, the need exists for a new, simpler and more economicsynthesis for this substance. Therefore, an object of the invention isto provide a synthesis of this type.

The object is solved according to the invention by providing a synthesisfor magnesium pyridoxal-5'-phosphate glutamate in whichpyridoxal-5'-phosphate is obtained as an intermediate product. Thepyridoxal-5'-phosphate accumulates in the form of an aqueous solutionfrom which the pyridoxal-5'-phosphate can be isolated if required.However, the obtained pyridoxal-5'-phosphate solution can also bereacted to the end product in an advantageous manner without isolationof the pyridoxal-5'-phosphate. Therewith, the steps required accordingto the state of the art for purification and drying of thepyridoxal-5'-phosphate are omitted.

A further point of the invention relates to the fact that, starting frompyridoxine, and especially an acid addition salt of pyridoxine, such aspyridoxine hydrochloride, pyridoxal-5'-phosphate is produced in a highyield according to a new method with p-phenetidyl pyridoxal andp-phenetidyl-pyridoxal-5'-phosphate as intermediate products.

Subject matter of the invention is a method for the synthesis ofmagnesium pyridoxal-5'-phosphate glutamate which is characterised inthat:

A) pyridoxine or an acid addition salt thereof is oxidised withmanganese(IV) oxide to pyridoxal;

B) pyridoxal is reacted with p-phenetidine under formation of theSchiff's base p-phenetidyl-pyridoxal;

C) p-phenetidyl-pyridoxal is selectively phosphorylated on the5'-hydroxymethyl group under formation ofp-phenetidyl-pyridoxal-5'-phosphate;

D) p-phenetidyl-pyridoxal-5'-phosphate is hydrolysed under formation ofan alkali metal salt of pyridoxal-5'-phosphate;

E) the alkali metal ions are removed in order to obtainpyridoxal-5'-phosphate;

F) pyridoxal-5'-phosphate is reacted with a reaction product of amagnesium alcoholate and L-glutamic acid; and

G) the formed magnesium pyridoxal-5'-phosphate glutamate is isolated.

A preferred embodiment of the invention is represented in the followingreaction scheme. ##STR1##

Molecular weight of the compounds.

    __________________________________________________________________________    manganese(IV) oxide                       MnO            86,9 g/mol    para-phenetidine   (7)                          C.sub.8 H.sub.11 NO                                     137,0 g/mol    para-phenetidyl-pyridoxal                       (4)                          C.sub.16 H.sub.18 N.sub.2 O.sub.3                                     286,0 g/mol    para-phenetidyl-pyridoxal-5'-phosphate                       (3)                          C.sub.16 H.sub.19 N.sub.2 O.sub.6 P                                     366,0 g/mol    pyridoxal          (5)                          C.sub.8 H.sub.9 NO.sub.3                                     167,0 g/mol    pyridoxal-5'-phosphate                       (2)                          C.sub.8 H.sub.10 NO.sub.6 P                                     247,0 g/mol    pyridoxal-5'-phosphate monohydrate                       (2')                          C.sub.8 H.sub.12 NO.sub.7 P                                     265,0 g/mol    pyridoxine hydrochloride                       (6)                          C.sub.8 H.sub.12 ClNO.sub.3                                     205,5 g/mol    magnesium pyridoxal-5'-phosphate                       (1)                          C.sub.3 H.sub.11 Mg.sub.2..sub.5 N.sub.2 O.sub.9                                     432,0 g/mol    glutamate    magnesium pyridoxal-5'-phosphate glutamate                       (1')                          C.sub.13 H.sub.15 Mg.sub.2..sub.5 N.sub.2 O.sub.11                          P          464,0 g/mol    dihydrate    __________________________________________________________________________

It has been shown to be advantageous to carry out the individualsynthesis steps discontinuously in suitable temperature and pHcontrolled units under light and/or air exclusion.

In order to be able to monitor the chemical reaction at any time, it ispossible to carry out in-process controls for example. In-processcontrols of this type can comprise analyses, such as HPLC analyses,which can be reliably carried out in a short amount of time (15 min).

Furthermore, it has been shown to be suitable to release the product ofeach step for further processing first after carrying out an analysisand a purity test.

The individual steps of the synthesis are illustrated in detail in thefollowing.

According to the invention, pyridoxine, preferably in form of an acidaddition salt such as pyridoxine hydrochloride (6), is first oxidised topyridoxal (5). A suitable oxidation agent is manganese(IV) oxide whichis suitably used in activated form. The oxidation is then preferablycarried out in sulfuric acid solution under controlled pH andtemperature conditions as well as under light exclusion. The degree ofreaction can be continuously examined by means of a suitable analyticalsystem (for example, HPLC). It is particularly preferred to stop thereaction as soon as the entire amount of pyridoxine was oxidised. Asopposed to known methods of oxidation of pyridoxine hydrochloride (6),the amount of oxidation side products formed can be diminished and theyield of pyridoxal (5) can be increased thereby.

The described oxidation of a pyridoxine acid addition salt to pyridoxal(5) with manganese(IV) oxide is typically carried out in the acidicrange without adherence to particular pH or temperature conditions.However, oxidation with manganese(IV) oxide is carried out in aparticularly advantageous manner at a constant pH value of 5,1 and aconstant temperature of 14° C. for further increasing the yield andminimising the amount of side products formed.

The reaction mixture can subsequently be subjected to processing forseparation, for example by filtration, of precipitated material such asreacted or non-reacted oxidation agent, for example manganese salts.Thereby, it is also possible to recover non-reacted oxidation agent,such as manganese(IV) oxide and newly add this later to the process.

Isolation of pyridoxal (5) can be carried out by the person skilled inthe art according to new methods without difficulty.

However, according to the invention, isolation is not required and theobtained pyridoxal solution can be directly used as an aqueous pyridoxalsolution in the next reaction step.

An aqueous solution of pyridoxal (5), preferably relating to thereaction solution obtained in the oxidation, is brought to a weaklyacidic pH value and preferably a pH value of 4.5. After that,p-phenetidine (7) is added preferably in slight excess. After conclusionof the reaction, the formed precipitate is separated (preferablyfiltered), for example by washing with water and/or an organic solvent,purified, and preferably dried, whereby p-phenetidyl-pyridoxal (4) isobtained.

Excess p-phenetidine (7) can be recovered. If ions originating from theoxidation agent, such as manganese(II) ions, are contained in thesolution obtained after the separation of the precipitate, these canalso be recovered by precipitation with the aid of lye for example.

The Schiff's base p-phenetidyl-pyridoxal (4) is subjected to a treatmentfor selective phosphorylation of the 5'-hydroxymethyl group underformation of p-phenetidyl-pyridoxal 5'-phosphate (3). Thephosphorylation occurs through treating p-phenetidyl-pyridoxal (4) witha suitable phosphorylation agent. For example, reaction withpolyphosphoric acid is suitable.

In this case, phosphorylation preferably occurs under controlledtemperature conditions (4 to 25° C.) as well as light exclusion. For anoptimal reaction course, it has been shown to be favourable to bring thedried p-phenetidyl-pyridoxal (4) to a particle size of ≦500 μm beforethe reaction. Furthermore, it is advantageous to mixp-phenetidyl-pyridoxal (4) and polyphosphoric acid as intimately aspossible for reaction, for example in a kneading unit. The degree ofreaction can be constantly examined by means of a suitable analyticalsystem (for example, HPLC). It is particularly preferred to stop thereaction as soon as the entire amount of p-phenetidyl-pyridoxal (4) isphosphorated.

According to a particularly preferred embodiment, polyphosphoric acidand p-phenetidyl-pyridoxal are used in the ratio of 4-6 parts to 1 part.

In the above described reaction,p-phenetidyl-pyridoxal-5'-polyphosphates form. Then, through thesubsequent addition of water and acid, a partial hydrolysis occurs underformation and precipitation of p-phenetidyl-pyridoxal-5'-phosphate (3).The precipitation can be promoted by addition of lye. The compoundobtained is purified in the customary manner, for example by washingwith water and/or an organic solvent. It can be dried and isolated, butis suitably processed as a moist precipitate for the purpose of furthersynthesis.

The phosphate accumulating due to the excess of polyphosphate can bereacted with calcium hydroxide to calcium phosphate and can beoptionally easily stored with low risk and/or further processed ordisposed.

p-phenetidyl-pyridoxal-5'-phosphate (3) is then hydrolysed to pyridoxal5'-phosphate (2) and p-phenetidine (7) by treatment with an aqueousalkali solution, preferably at a pH value of more than 12.5. The formedp-phenetidine and the alkali metal salt of pyridoxal 5'-phosphate can beseparated according to any suitable method. p-phenetidine (7) can beseparated from the aqueous solution by extraction with a suitableorganic solvent, for example an aliphatic or aromatic hydrocarbon suchas toluol.

Alternatively, the p-phenetidine (7) can be separated according to apreferable embodiment by means of a liquid-liquid separator unit. Aseparation of this type is more time-saving, more ecological, moreefficient, easier and therewith more economic than the extraction.

The p-phenetidine (7) recovered in this reaction step can be purified bymeans of distillation for example and then newly used in the firstreaction step.

The alkali metal salt of pyridoxal-5'-phosphate (2) can be precipitatedfrom the obtained aqueous solution by concentration. The precipitate canbe further purified by washing or recrystallization for example.

In order to produce magnesium pyridoxal-5'-phosphate glutamate (1) fromthe alkali metal salt of pyridoxal-5'-phosphate (2), the alkali metalions must first be removed. For this purpose, any suitable method can beemployed. For example, the obtained aqueous alkali metal salt solutionof pyridoxal-5'-phosphate can be subjected to an ion exchange treatment.The solution obtained thereby can --after optional concentration--bedirectly used for synthesis of magnesium pyridoxal-5'-phosphateglutamate (1). It is also possible to isolate freepyridoxal-5'-phosphate (2) from the aqueous solution and to purify thecompound according to customary methods known to the person skilled inthe art. For the next synthesis step, an aqueous solution of previouslyisolated and purified pyridoxal -5'-phosphate (2) can be used in thiscase.

The aqueous solution of pyridoxal-5'-phosphate (2) is added to asolution which was obtained by reaction of a magnesium alkolholate withwater and addition of glutamic acid (8). magnesium ethylate (9) isparticularly preferred as a magnesium alcoholate because ethanol is thenreleased by the reaction which does not encumber the pharmaceuticalquality of the produced active ingredient with any undesired residues.The mixture is then added to a suitable solvent, preferably ethanol. Thereaction is preferably carried out in the cold under air and lightexclusion in as much as this is conducive to no side products forming.Magnesium pyridoxal-5'-phosphate glutamate (1) can be separated from thesuspension by filtration for example. The product obtained in thismanner can be purified by washing and/or recrystallization andsubsequently dried.

By using magnesium ethylate and ethanol, a solution is obtained afterseparation of magnesium pyridoxal-5'-phosphate glutamate (1) whichconsists of water and ethanol. The solution can be simply disposed of orcan be separated by distillation into its components.

In the following, the invention is illustrated further by an example. Inthis example, the synthesis of magnesium pyridoxal-5'-phosphateglutamate (1) occurs with the aid of the following equipment:

a) temperature controlled mixing vessel (0 to 100° C.) with pHmonitoring and metering unit;

b) temperature controlled kneading unit (0 to 100° C.) with stronggearing in the low rotational speed range;

c) extraction unit (aqueous/organic) and/or liquid-liquid separator;

d) cation exchanger unit;

e) filtration unit and/or solid-liquid separator; and

f) drying unit.

HPLC analyses were conducted under the following conditions:

HPLC Conditions

1. System

a) chromatographic system:

Shimadzu-unit comprising: LC-10AS liquid chromatograph; SIL-10A AutoInjector; SPD-10AV (UV-VIS; spectrophotometric detector); CBM-10ACommunications Bus Module; CLASS-LC10 Software; PC 486, 40 Mhz; Desk Jet560 C Hewlett Packard.

Stationary Phase (column)

Hypersil ODS 5 μm 250×4.6 mm with pre-column cartridge 20×4.6 mm

Mobile Phase (eluent)

aqueous 0.001 m KH₂ PO₄ solution with H₃ PO₄ adjusted to pH=3.

    ______________________________________    flow                1.5    ml/min    injection           5      μl    detection           295    nm    analytical time     15     min    ______________________________________

b) preparation: a small (for example drop-size) residual amount of asimilar size as possible from the reaction mixture (for example twodrops with liquid) is dissolved in five drops 10% sulfuric acid.

HPLC conditions

2. System

a) chromatographic system:

Shimadzu-unit comprising: LC-10AS liquid chromatograph; SIL-10A AutoInjector; SPD-10AV (UV-VIS; spectrophotometric detector); CBM-10ACommunications Bus Module; CLASS-LC10Software; PC 486, 40 Mhz; Desk Jet560 C Hewlett Packard.

Stationary Phase (column)

Hypersil ODS 5μm 250×4.6 mm with pre-column cartridge 20×4.6 mm

Mobile Phase (eluent): 5% B

A≡aqueous 0.001 M KH₂ PO₄ solution with H₃ PO₄ adjusted to pH=3.

B≡acentonitrile

    ______________________________________    flow                1.5    ml/min    injection           5      μl    detection           295    nm    analytical time     10     min    ______________________________________

b) preparation: a small (for example drop-size) residual amount of asimilar size as possible from the reaction mixture (for example twodrops with liquid) is dissolved in five drops 10% sulfuric acid.

PRODUCTION EXAMPLE 1st Step

Production of p-phenetidyl-pyridoxal (4) from pyridoxine hydrochloride(6)

100 g pyridoxine hydrochloride (6) are dissolved in 1500 g water andadjusted to a pH of 5.10 with cold 4N sodium hydroxide solution. Theobtained solution is brought to a temperature of 14° C. and added to 75g activated manganese(IV) oxide under strong stirring.

The reaction starts immediately after manganese dioxide addition and thepH value increases if it is not permanently adjusted. Through additionof 100% sulfuric acid in portions, the pH value of the reactionsuspension is held constant at 5.10±0.10 by means of a constant pHconstant holder under intensive stirring and temperature control (14±2°C.). The course of the reaction is followed by means of HPLC (system 1)in the form of in-process controls. After approximately ten hours, theentire amount of pyridoxine hydrochloride was converted into pyridoxal.After that, the excess manganese dioxide--non-reacted and contaminatedwith pyridoxal--is filtered, washed three times with approximately 300 gdeionized water and dried at 120° C. 25 g of manganese dioxide isrecovered which can be used again. The combined aqueous solutions arefirst subjected to a membrane filtration (0.8 μm) and then adjusted topH 4.5 with 10% sulfuric acid. Then, 80 g freshly distilledpara-phenetidine is added in a portion to the pyridoxal solution undervery intensive stirring. Thereby, the pH value of the reaction mixtureincreases to about 4.7 and the reaction product abruptly precipitates.This is further stirred for half an hour and subsequently filtered. Thepara-phenetidine containing therein is recovered by means of aliquid-liquid separator from the filtrate which is to be disposed.

The precipitate is suspended three times each in 700 g deionized waterand filtered. Subsequently, it is suspended and filtered by means of anUltra-Turax in 700 g n-heptane. The n-heptane filtrate is redistilledand the collected para-phenetidine residues are stored until conclusionof the third step.

The residue obtained in this manner is dried at 120° C. and sifted to aparticle size of ≦500 μm. 124.8 g (=89.7% theoretical yield) of a fine,very light orange-yellow powder of para-phenetidyl-pyridoxal (4) isobtained.

2nd Step

Production of para-phenetidyl-pyridoxal-5'-phosphate (3) frompara-phenetidyl-pyridoxal (4).

1000 g of Polyphosphoric acid are placed into a temperature controlledkneading unit and cooled to 10° C. 250 g para-phenetidyl-pyridoxal (4)are slowly applied in small portions to the Polyphosphoric acid underintensive cooling. The Schiff's base of the pyridoxal slowly dissolvesin the phosphorylation reagent under red coloration. The reaction heatformed thereby must be intensively carried off in such a manner that thetemperature of the reaction mixture does not exceed 25° C. A temperatureincrease to about the double of this leads to approximately 20%additional loss of yield. After the entire para-phenetidyl-pyridoxalmass was applied, the reaction mixture is further stirred underintensive cooling (≦25° C.) overnight. On the following day, no morepara-phenetidyl-pyridoxal is to be identified by means of an in-processcontrol with the aid of HPLC (system 1). The phosphorylation step isconcluded therewith. Subsequently, approximately 2125 g ice are addedinto the mixing chamber and the reaction suspension is stirred furtherunder intensive cooling for three hours. A homogeneous mustard-colouredmass forms while doing so. It is slowly added to 75 g 95-97% coldsulfuric acid, homogenised and heated for approximately 30 min at 80°C., wherein the polyphosphates are hydrolysed. The hydrolysis isfollowed by means HPLC (system 2), whereby the exact moment of the stopof hydrolysis is recognised (based on the complete disappearance of thePolyphosphate peak). The end of hydrolysis is brought about by rapidcooling of the reaction solution to 10° C. Thereby, the phosphorylatedproduct begins to precipitate. Then, approximately 7500 g of 2N sodiumhydroxide solution pre-cooled to approximately 10° C. are slowly addedunder intense stirring and cooling until the pH value of the reactionsuspension is 2.10. The orange-brown reaction product precipitatedthereby is filtered well and washed three times each with approximately500 g deionized water. The well filtered precipitate is directly usedfor the third step of the synthesis.

Nevertheless, should the product of this step be dried, this can occurat 105° C. 263.4 g (=82.3% theoretical yield) of an orange-brown finepowder of para-phenetidyl-pyridoxal-5'-phosphate (3) would be obtained.

3rd Step

Production of pyridoxal-5'-phosphate (2) frompara-phenetidyl-pyridoxal-5'-phosphate (3).

100 g and/or the corresponding amount of the moist precipitate ofpara-phenetidyl-pyridoxal-5'-phosphate (3) obtained in the second stepare added to 700 g 2N sodium hydroxide solution. The Schiff's base ishydrolysed to pyridoxal-5'-phosphate and para-phenetidyl at pH ≧12.5. Inthe case that the above given amount of lye is not sufficient in orderto obtain a pH ≧12.5--on account of the moist precipitate --thenecessary amount of additional 2N sodium hydroxide solution is used. Byextracting three times each with 300 g toluol, the para-phenetidine isremoved from the solution. The toluol solution is redistilled. As analternative to toluol extraction, an organic-aqueous-separator forcontinuous operation can be used for the separation of para-phenetidine.The collected para-phenetidine residues from the first and third stepscan be purified by means of distillation and newly used.

The pyridoxal-5'-phosphate is present at the end of this separationoperation as a sodium salt in the aqueous solution. By means of 800 mlcation exchanger, for example Amperlite Type IR-120 (1.9 mVal/ml), thesodium ion is removed from pyridoxal-5'-phosphate. A pure aqueoussolution of pyridoxal-5'-phosphate is obtained. 2500 ml eluate contain57-63 g (78.6-86.9% theoretical yield) pyridoxal-5¹ -phosphate (2). Thisis concentrated under vacuum at max. 40° C. to approximately 500 ml andused for MPPG production. The Pyridoxal-5'-phosphate synthesised in thismanner can also be isolated at this point (new synthesis ofpyridoxal-5'-phosphate).

4th Step

Production of magnesium pyridoxal-5'-phosphate glutamate (1) frompyridoxal-5'-phosphate (2).

540 g deionized water are placed into a reaction vessel and cooled to0-3° C. Under nitrogen atmosphere and light exclusion, 62.58 g magnesiumethylate (9) are added and brought into solution by stirring. Thereby,the temperature increases to approximately 10° C. This is cooled to 3°C., 31.85 g glutamic acid (8) are added, and this is stirred for afurther 10 min.

Then, 500 ml pyridoxal-5'-phosphate solution of the third step (≡11.6%solution) is added in small portions within 60-90 min. to the magnesiumglutamate solution just produced. Thereby, attention is paid that the pHvalue of the solution remains within the range 8-9. Subsequently, thisis further stirred for 3-5 hours and the resulting solution is filtered.

2000 ml ethanol is placed into a mixing vessel and cooled to 0° C. undernitrogen atmosphere and light exclusion. Subsequently, the solutionproduced above is added drop-wise under stirring. The suspensionattained in this manner is further stirred for approximately 15 hours,then filtered, and washed with a total of 200 ml ethanol. After that,the entire product is suspended in approximately 100 ml ethanol andnewly filtered. The ethanol-moist product is dried in a drying cabinetat max. 60° C. 94.5-99.5 g (94-99% theoretical yield) of magnesiumpyridoxal-5'-phosphate glutamate (1) is obtained.

We claim:
 1. A method for the production of magnesiumpyridoxal-5'-phosphate glutamate comprising:A) oxidizing pyridoxine oran acid addition salt thereof with manganese(IV) oxide to pyridoxal; B)reacting pyridoxal with p-phenetidiene to form p-phenetidyl-pyridoxal;C) phosphorylating p-phenetidyl-pyridoxal to formp-phenetidyl-pyridoxal-5'-phosphate; D) hydrolyzingp-phenetidyl-pyridoxal-5'-phosphate to form an alkali metal salt ofpyridoxal-5'-phosphate; E) removing the alkali metal ions to obtainpyridoxal-5'-phosphate; F) reacting pyridoxal-5'-phosphate with areaction product of a magnesium alcoholate and L-glutamic acid; and G)isolating magnesium pyridoxal-5'-phosphate glutamate.
 2. A methodaccording to claim 1, wherein the acid addition salt of pyridoxine usedin step A is pyridoxine hydrochloride.
 3. A method according to claim 1wherein the oxidation in step A is carried out with activatedmanganese(IV) oxide in sulfuric acid solution at a pH value of about 5.1and a constant temperature of about 14° C.
 4. A method according toclaim 1 wherein a pyridoxal containing reaction solution of theoxidation in step A is used in step B.
 5. A method according to claim 1wherein the phosphorylation in step C is carried out by reaction ofp-phenetidyl-pyridoxal with polyphosphoric acid.
 6. A method accordingto claim 5, wherein polyphosphoric acid and p-phenetidyl-pyridoxal areused in a ratio of about 4 parts polyphosphoric acid to about 1 partp-phenetidyl-pyridoxal.
 7. A method according to claim 1 wherein analkali hydroxide is added at step D in an amount effective forhydrolyzing p-phenetidyl-pyridoxal.
 8. A method according to claim 1wherein the alkali metal ions in step E are removed by an ion exchanger.9. A method according to claim 1 wherein the magnesium alcoholate instep F is magnesium ethylate.
 10. A method for the production ofpyridoxal-5'-phosphate comprising:A) oxidizing pyridoxine or an acidaddition salt thereof with manganese(IV) oxide to pyridoxal; B) reactingpyridoxal with p-phenetidiene to form p-phenetidyl-pyridoxal; C)phosphorylating p-phenetidyl-pyridoxal to formp-phenetidyl-pyridoxal-5'-phosphate; D) hydrolyzingp-phenetidyl-pyridoxal-5'-phosphate to form an alkali metal salt ofpyridoxal-5'-phosphate; E) removing the alkali metal ions to obtainpridoxal-5'-phosphate; F) isolating and drying thepyridoxal-5'-phosphate obtained in step E.